Mg2+-imidazole-catalyzed self-condensation of malonyl thioesters: Getting tuned for biomimetic polyketide synthesis?

Article abstract of DOI:10.3390/61100845

We report that a subtle balance of carbanion reactivity, leaving group activation, and pK_a of the catalyst is required for efficient self-condensation of thiomalonates to thioacetoacetates in up to 71% yield under "biomimetic" conditions originally proposed by Kobuke and Yoshida (Tetrahedron Lett. 1978, 19, 367).

Full text of DOI:10.3390/61100845

Molecules 2001, 6, 845-851
molecules
ISSN 1420-3049
http://www.mdpi.org
Mg2+-Imidazole-Catalyzed Self-Condensation of Malonyl
Thioesters: Getting Tuned for Biomimetic Polyketide Synthesis?
Naomi Sakai, Nathalie Sordé and Stefan Matile*
Department of Organic Chemistry, University of Geneva, CH-1211 Geneva 4, Switzerland. FAX:
(+41) 22 328 7396.
* Author to whom correspondence should be addressed; E-mail: stefan.matile@chiorg.unige.ch
Received: 3 September 2001; in revised form 15 October 2001 / Accepted: 16 October 2001 /
Published: 31 October 2001
Abstract: We report that a subtle balance of carbanion reactivity, leaving group activation,
and pKa of the catalyst is required for efficient self-condensation of thiomalonates to
thioacetoacetates in up to 71% yield under “biomimetic” conditions originally proposed by
Kobuke and Yoshida (Tetrahedron Lett. 1978, 19, 367).
Keywords: Bioorganic chemistry, Claisen condensation, enzyme mimics, polyketide
synthesis.
The Claisen condensation of malonyl thioesters is one of the central processes in the biosynthesis of
polyketide natural products [1]. Although the diverse enzymes that catalyze this remarkable
biooligomerization are increasingly well understood and extensively exploited in modern
biotechnology [2-4], synthetic catalysts for similarly controlled oligomerization of malonyl thioesters
in enzyme-free systems do not exist. Kobuke and Yoshida have, however, demonstrated more than
twenty years ago that Claisen condensation of n-butyl thiomalonate 1a and phenyl thioacetate 2e can be
catalyzed by imidazole and magnesium cations in THF at room temperature to give n-butyl
thioacetoactate 3a in 87 h and 60% yield (Scheme 1) [5]. These Kobuke-Yoshida (KY) conditions
[5,6] contrast sharply with the harsher conditions required in other model systems for polyketide
synthesis [7-9] but not polyketide cyclization [10-12].

Molecules 2001, 6
846
Scheme 1
imidazole
O
O
O
O
O
SnBu
SPh
SnBu
Mg(OAc)
HO
2
THF, RT
1a
2e
3a
Results and Discussion
The original KY-conditions are of potential significance for the construction of artificial polyketide
synthases because many histidine-rich organic architectonics with esterase and/or phosphatase activity
have been elegantly devised over the past two decades [13-15]. Original KY-conditions are, however,
incompatible with the Claisen self-condensation required for oligomerizations leading to polyketides
(Scheme 2). Specifically, self-condensation of n-butyl thiomalonate 1a or phenyl thioacetate 2e was
impossible because of poor leaving group (LG) ability of n-butylthiolate or lack of access to activated
carbanion intermediates via decarboxylation, respectively [5]. Here we report that Mg2+-imidazole-
catalyzed thiomalonate dimerization is possible in up to 71% yield under refined conditions by precise
fine tuning of the properties of carbanion intermediate, thiolate leaving group, and catalyst, and show
that future applicability of this approach toward biomimetic polyketide synthesis is nevertheless
problematic.
Scheme 2
cat
O
O
O
O
O
δ
X
R
X
X
1
Mg(OAc)
THF
HO
S
2
S
+
S
R
R
R
R
1a-g
3a-g: R = H
2a-g
1
TMSCHN
2
4a-g: R = CO H
1
2
cat
Mg(OAc)
THF
O
O
O
O
2
O
S
oligomers?
5c
To elucidate the subtle balance between leaving group and carbanion activation needed for
thiomalonate dimerization under KY-conditions, we prepared substrates 1a-f with systematically
varied leaving groups (LG) (Scheme 2, Table 1). Reduced Hammett σp [16] (corresponding with one
exception to the pKa of the employed thiophenols [17,18]) should reduce LG-activation and increase
the reactivity of carbanion intermediates, which can be seen as up-field shifts of the α-hydrogens
(Table 1). Indeed the rate of substrate consumption was inversely related to the Hammett σp.

Molecules 2001, 6
847
However, highly stabilized carbanions did not further react with the electrophiles, thus dominant
formation of decarboxylation products 2e-2g resulted. Thiomalonate self-condensation giving rise to
thioacetoacetates 3b-3f in up to 37% yield for 3c occurred only at intermediate activation of both
carbanion and LG.
Table 1. Claisen self-condensation of thio(methyl)malonates 1a-g in THF at
room temperature in presence of Mg(OAc) and imidazole.
2
c
a
b
d
a
σ
p pK (LG) t (h)
entry Substrate
R
δ (ppm)
3 (%)
X
a
1
2
3
4
5
6
7
1a
1b
1g
1c
1d
1e
1f
H
H
3.62
3.65
-
~11
-
0
10
0
-
9.43
96
72
17
2
CH 3.79 -0.28 7.06
OCH3
3
H
H
H
H
3.69 -0.28 7.06
3.70 -0.14 7.08
37
30
14
5
OCH3
CH3
H
3.72
3.73
0.00
0.24
6.81
6.53
1
Cl
1
aCompare Scheme 2. bChemical shift δ of the α-proton(s) in the 1H NMR spectra of
1a-g. cpKa of the conjugate acid of the thiolate leaving groups (LG). dYields refer to
NMR- and HPLC-pure products isolated after reaction time t needed for substrate
consumption.
The most promising thiomalonate 1c was studied in more detail. Separation of the product mixture
by reverse-phase HPLC was possible on analytical YMC-Pro-C8-columns applying a linear solvent
gradient of water / CH3CN (0.1% TFA) = 20% - 100% over 20 min. Only one additional product with
a retention time Rt = 5.22 min similar to that of substrate 1c (Rt = 4.94 min) was observed besides the
expected acetoacetate 3c (Rt = 5.90 min), 4-thioanisole (Rt = 6.31 min) and 4-methoxyphenyl
thioacetate 2c (Rt = 6.31 min). This new product decomposed easily and could not be fully purified.
However, in-situ methylation with trimethylsilyl diazomethane (TMSCHN2, [19]) in toluene-methanol
gave a stable product with m/z = 319 in the ESI-MS that was consistent with [M + Na]+ expected for
methyl ester 5c. The 1H-NMR spectrum of 5c revealed about 50%-conjugation of the enol ether with
both carbonyl groups. This derivatization demonstrated that the new unstable product formed from 1c
under KY-conditions is carboxylate 4c. Identification of 4c allowed us to assign all new resonances
appearing in 1H-NMR spectra recorded during the course of a reaction in THF-d8 to either 3c, 4c, 2c or
acetylimidazole.
The satisfactory outcome with substrate 1c encouraged us to study the influence of additional
parameters. Thiomethylmalonate 1g gave decarboxylation product 2g only, probably due to the steric
effect of the methyl group on the α-position (Table 1). Replacement of Mg2+ by other divalent cations
such as Zn2+, Cu2+, Ca2+, or Ba2+ under otherwise original KY conditions led to an increase in acetate

Molecules 2001, 6
848
(2a-2f) and/or hydrolyzed products rather than Claisen self-condensation. The rate of substrate
consumption decreased with decreasing pKa of the imidazole catalyst [20] (Table 2). The yield of
Claisen products 3c and 4c, however, increased from imidazole (36%) over benzimidazole (47%) to
71% with 8-nitrobenzimidazole. Further decrease in catalyst pKa gave 51% for 4(5)-nitroimidazole.
This identified pKa ≈ 3 as optimum catalyst pKa for Claisen self-condensation under these conditions
(Table 2).
Table 2. Claisen self-condensation of thiomalonate 3 in THFd at
room temperature in presence of Mg(OAc)2 and the indicated
imidazole catalyst.
a
cata
entry
pKa
t (h)
3 (%)b 3a+3b (%)c
N
36 (22 + 14)
12
3
1
2
6.95
5.40
NH
N
47 (14 + 33)
18
46
NH
O2N
O2N
N
71 (34 + 37)
5
48
72
3
NH
3.05
3.05
4d
N
35 (25 + 10)
30
NH
O2N
NH
N
51 (18 + 33)
17
92
5
1.50
b
aImidazole catalysts of different pKa. Yields refer to remaining substrate at time
t according to 1H-NMR-spectra of the reaction mixture. cYields refer to NMR-
and HPLC-pure products isolated, yields in parenthesis give the relative
distribution between Claisen products 3a and 3b in RP-HPLC of product mixtures
after reaction time t (Scheme 2). dCHCl3/18-crown-6 9:1 instead of THF.
Replacement of THF with other solvents except dioxane and addition of more than 10% water
inhibited self-condensation. Kobuke and Yoshida’s original implication that ether-coordination to
Mg2+ is essential for catalysis was further partially supported by slow appearance of up to 35% Claisen
products 3c and 4c in CHCl3 containing 10% 18-crown-6 with 8-nitrobenzimidazole as catalyst (Table
2, entry 4). The reaction did, however, not proceed in the “ether-rich” micelles formed by 10% Triton
X-100 in water, also when N-acetylhistidine was used instead of imidazole or 8-nitrobenzimidazole.
No indications for further reaction of thiomalonate 1c with Claisen products 3c or 4c were found.
This suggests that Aldol and Claisen reaction with β-ketone and thioester for formal continuation along
the terpenoid and polyketide pathway [1], respectively, are not possible under these conditions. Novel
approaches toward transient deactivation of the β-ketone in acetoacetates required for polyketide
synthesis under these conditions are under investigation. Preliminary studies with “additives” such as
TMSCHN2, TES-Cl, n-butylamine, p-methoxyaniline and phenylhydrazine [21] were not successful.

Molecules 2001, 6
849
Conclusions
In summary, we have found that Claisen self-condensation of thiomalonates to thioacetoacetate
under “biomimetic” conditions is dependent on a subtle balance of carbanion and leaving group
activation and the pKa of the imidazole catalyst. These findings demonstrate that future development
of more refined artificial “metallo-Claisenases” is possible along the route originally proposed by
Kobuke and Yoshida [5]. The possibility to expand Mg2+-imidazole-catalyzed Claisen self-
condensation under these conditions toward polyketide synthesis remains, however, questionable.
Acknowledgments
We thank Prof. J. Yoshida (Kyoto University) for fruitful discussions, A. Pinto, J.-P. Saulnier, the
group of Prof. F. Gülaçar, and Dr. H. Eder for NMR, MS, and elemental analyses, respectively, and the
Swiss NSF (21-57059.99 and National Research Program “Supramolecular Functional Materials”
4047-057496) for financial support.
Experimental
General
Synthetic reagents were purchased from Fluka or Acros. Column chromatography was carried out
on silica gel 60 (Fluka, 40-63 mm). Analytical thin layer chromatography (TLC) and preparative thin
layer chromatography were performed on silica gel 60 (Fluka, 0.2 mm) and silica gel GF-2 (Aldrich, 1
mm), respectively. Reverse Phase column chromatography was performed with Silicagel 100 C18-
1
Reverse Phase. H- and 13C-NMR spectra were recorded on Bruker 400 MHz Spectrometer. ESI-MS
were performed on a Finnigan MAT SSQ 7000. HPLC was carried out using a YMC Pro-C8 (4 x 50
mm) prepacked column. Anhydrous THF and ethyl ether were distilled over Na and benzophenone.
Thiomalonate 1c: General procedure for preparation of thiomalonate esters
Thiomalonate esters were prepared following the procedure in reference [5]. Namely, to a solution
of malonylchloride (1.6 mL, 16 mmol) in dry ether (20 mL) 4-methoxybenzenethiol (2.0 mL, 16
mmol) was added at room temperature (rt) under nitrogen atmosphere. After stirring for 3h, saturated
aqueous NaHCO3 solution was added to the reaction mixture. The ether layer was discarded, and the
aqueous layer was further washed with EtOAc. After acidification of the aqueous layer, the product
was obtained by extraction (EtOAc), washing (brine), drying (Na2SO4) and evaporation under reduced
pressure. The crude product was purified by silica gel column chromatography (acetone) then by
recrystalization (dichloromethane) to give pure title compound as colorless crystals (1.7 g, 45 %): mp

Molecules 2001, 6
850
89-90 ºC; 1H-NMR (CDCl3) δ 9.80 (br.s, 1 H), 7.36 (d, J = 6.7 Hz, 2 H), 6.95 (d, J = 6.7 Hz, 2 H), 3.83
13
(s, 3 H), 3.69 (s, 2 H); C-NMR (CDCl3) δ 191.0 (s), 171.0 (s), 161.0 (s), 136.1 (2 x d), 117.0 (s),
115.1 (2 x d), 55.4 (q), 48.0 (t).
Acetoacetate 3c: General procedure for Claisen condensation
To a solution of 4-methoxyphenyl thiomalonate (1c, 20 mg, 0.088 mmol) in THF (1 mL),
Mg(OAc)2•4H2O (10 mg, 0.047 mmol) and imidazole (6 mg, 0.088 mmol) were successively added.
The mixture was stirred for 17h at rt, and then it was diluted with CH2Cl2, washed with 1M HCl and
brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by
column chromatography (1:1 to 0:1 petroleum-ether/CH2Cl2) to give pure acetoacetate 3c as a colorless
1
oil (3.7 mg, 37 %): H-NMR (CDCl3) δ 7.37 (d, J = 8.8 Hz, 0.6 H), 7.32 (d, J = 8.8 Hz, 1.4 H), 6.94
(d, J = 8.8 Hz, 2 H), 5.47 (s, 0.3 H), 3.82 (s, 3 H), 3.73 (s, 1.4 H), 2.27 (s, 2.1 H), 1.94 (s, 0.9 H).
Methyl ester 5c
The crude product obtained following the general procedure for Claisen condensation starting from
1c (213 mg) was fractionated by column chromatography (ODS, 40 to 100 % CH3CN in 0.1 % aqueous
TFA). The fractions containing 4c were concentrated briefly and then extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure to
give crude product (32 mg) consisting of 1c and 4c. The product mixture obtained was dissolved in
toluene (0.5 mL) and methanol (0.5 mL), and TMSCHN2 (2 M in hexane, 0.3 mL) was added. After
stirring for 30 min, the mixture was concentrated under reduced pressure and purified by preparative
TLC (CH2Cl2, Rf 0.26) to give pure 5c (3.5 mg, 1.2%): 1H-NMR (CDCl3) δ 7.36 (d, J = 8.9 Hz, 2 H),
6.94 (d, J = 8.9 Hz, 0.8 H), 6.93 (d, J = 8.9 Hz, 1.2 H), 5.62 (s, 0.4 H), 5.23 (s, 0.6 H), 4.18 (s, 1.2 H),
3.83 (s, 1.2 H), 3.82 (s, 1.8 H), 3.80 (s, 0.8 H), 3.75 (s, 1.2 H), 3.71 (s, 1.8 H), 3.70 (s, 1.8 H), 3.67 (s,
1.2 H); ESI-MS (CH2Cl2 + TFA) m/z 334.9 (8.25) [M + K]+, 319.0 (100) [M + Na]+, 157.4 (31.9) [M -
MeOC6H4S]+.
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